Studies of the Silencing of Baculovirus DNA Binding Protein

Quadt, Ilja; Lent, J.W.M. van; Knebel-Mörsdorf, Dagmar

doi:10.1128/jvi.02768-06

Cited by 12 publications

(7 citation statements)

References 25 publications

(24 reference statements)

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“…RNAi is an invaluable tool for defining functions of virus and host genes in lepidopteran and dipteran cells (18,25,34,48,54,58). The success of RNAi-mediated inhibition of virus gene expression is due in part to the efficiency with which in vitro-synthesized double-stranded RNA (dsRNA) enters cultured insect cells and subsequently engages the host's RNA silencing machinery (reviewed in reference 33).…”

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confidence: 99%

Transactivator IE1 Is Required for Baculovirus Early Replication Events That Trigger Apoptosis in Permissive and Nonpermissive Cells

Schultz

Wetter

Fiore

et al. 2009

J Virol

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Immediate early viral protein IE1 is a potent transcriptional activator encoded by baculoviruses. Although the requirement of IE1 for multiplication of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is well established, the functional roles of IE1 during infection are unclear. Here, we used RNA interference to ablate IE1, plus its splice variant IE0, and thereby define in vivo activities of these early proteins, including gene-specific regulation and induction of host cell apoptosis. Confirming an essential replicative role, simultaneous ablation of IE1 and IE0 by gene-specific double-stranded RNAs inhibited AcMNPV late gene expression, reduced yields of budded virus by more than 1,000-fold, and blocked production of occluded virus particles. Depletion of IE1 and IE0 had no effect on early expression of the envelope fusion protein gene gp64 but abolished early expression of the caspase inhibitor gene p35, which is required for prevention of virus-induced apoptosis. Thus, IE1 is a positive, gene-specific transactivator. Whereas an AcMNPV p35 deletion mutant caused widespread apoptosis in permissive Spodoptera frugiperda cells, ablation of IE1 and IE0 prevented this apoptosis. Silencing of ie-1 also prevented AcMNPV-induced apoptosis in nonpermissive Drosophila melanogaster cells. Thus, de novo synthesis of IE1 is required for virus-induced apoptosis. We concluded that IE1 causes apoptosis directly or contributes indirectly by promoting virus replication events that subsequently trigger cell death. This study reveals that IE1 is a gene-selective transcriptional activator which is required not only for expedition of virus multiplication but also for blocking of its own proapoptotic activity by upregulation of baculovirus apoptotic suppressors.

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confidence: 99%

Transactivator IE1 Is Required for Baculovirus Early Replication Events That Trigger Apoptosis in Permissive and Nonpermissive Cells

Schultz

Wetter

Fiore

et al. 2009

J Virol

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“…Several groups have successfully taken advantage of the RNAi technique to silence baculovirus genes in insect cells (Ikeda et al, 2004;Means et al, 2003;Quadt et al, 2007). Furthermore, RNAi experiments have been performed successfully in S. litura insects (Rajagopal et al, 2002).…”

Section: Resultsmentioning

confidence: 99%

Functional analysis of the putative antiapoptotic genes, p49 and iap4, of Spodoptera litura nucleopolyhedrovirus with RNAi

Qian

Lin

Feng

et al. 2008

Journal of General Virology

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A homology search of a public database revealed that Spodoptera litura nucleopolyhedrovirus (SpltNPV) possesses two putative, antiapoptotic genes, p49 and inhibitor of apoptosis 4 (iap4), but their function has not been investigated in its native host cells. In the present study, we used RNA interference (RNAi) to silence the expression of Splt-iap4 and Splt-p49, independently or together, to determine their roles during the SpltNPV life cycle. RT-PCR analysis and Western blot analysis showed the target gene expression had been knocked out in the SpltNPV-infected SpLi-221 cells after treatment with Splt-p49 or Splt-iap4 double-stranded RNA (dsRNA), respectively, confirming that the two genes were effectively silenced. In SpltNPV-infected cells treated with Splt-p49 dsRNA, apoptosis was observed beginning at 14 h, and almost all cells had undergone apoptosis by 48 h. In contrast, budded virus production and polyhedra formation progressed normally in infected cells treated with Splt-iap4 dsRNA. Cell viability analysis showed that Splt-IAP4 had no synergistic effect on the inhibition of apoptosis of SpLi-221 cells induced by SpltNPV infection. Interestingly, after Splt-iap4 dsRNA treatment, cells did not congregate like those infected with SpltNPV in the early infection phase, implying an unknown role of baculovirus iap4. Our results determine that Splt-p49 is necessary to prevent apoptosis; however, Splt-iap4 has no antiapoptotic function during SpltNPV infection. INTRODUCTIONBaculoviruses are classified as a group of arthropodspecific viruses with rod-shaped nucleocapsids, and their genomes consist of a circular double-stranded DNA molecule of about 80-180 kbp (Theilmann et al., 2005). The family Baculoviridae comprises the genera Nucleopolyhedrovirus (NPV) and Granulovirus (GV) (Theilmann et al., 2005). Baculoviruses usually have limited host ranges and recent studies suggest that apoptosis plays a role in host range restriction (Zhang et al., 2002;Clarke & Clem, 2003;Feng et al., 2007).Apoptosis is a type of programmed cell death which is characterized frequently by nuclear condensation and fragmentation, accompanied by vigorous blebbing of the cytoplasm membrane (Kerr et al., 1972;Wyllie et al., 1980). The mechanism of apoptosis is evolutionarily conserved. A wide range of apoptotic stimuli, such as actinomycin D, UV light, virus infection etc., can trigger a family of cysteine protease proteins (caspases) to promote cell death (Roy et al., 1997;Thornberry & Lazebnik, 1998). All caspases are catalytically inactive zymogens and must undergo proteolytic activation during apoptosis. The caspases are generally divided into two classes: the initiator caspases and the effector caspases. Different apoptotic stimuli trigger the activation of the initiator caspases, which then cleave and activate the effector caspases (Riedl & Shi, 2004).Apoptosis represents an important virus-host interaction process which probably influences viral pathogenesis. As an antiviral response in multicellular organisms, apoptosis can limit ...

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“…The reason for the possession of two SSBs, LEF-3 and DBP, by many baculoviruses is not clear. Although both SSBs are required for the production of viable virions, DBP is not required for viral DNA synthesis (Vanarsdall et al, 2007;Quadt et al, 2007). However, the knockout of the dbp gene decreases the yield of nascent viral DNA and appears to prevent the production of mature full-size viral genomes (Vanarsdall et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

“…Upon fractionation of nuclei in aqueous solutions, both LEF-3 and DBP are found in a fraction of proteins that tightly binds to chromatin (Vanarsdall et al, 2007). In cells infected with AcMNPV, DBP is essential for production of viable virions, but in contrast to LEF-3, it is not required for synthesis of viral DNA or expression of viral genes (Vanarsdall et al, 2007;Quadt et al, 2007). However in the absence of a functional dbp gene, the virogenic stroma appeared to be absent and the yield of viral DNA was substantially decreased and no full-size viral genomes were produced (Vanarsdall et al, 2007) indicating that DBP may participate in the formation of the virogenic stroma and stabilize nascent viral DNA or participate in processing of replicative intermediates.…”

Section: Introductionmentioning

confidence: 99%

Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus

2008

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DNA-binding protein (DBP) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed as an N-terminal His(6)-tag fusion using a recombinant baculovirus and purified to near homogeneity. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein, LEF-3, for binding sites. DBP binding protected ssDNA against hydrolysis by a baculovirus alkaline nuclease AN/LEF-3 complex. Partial proteolysis by trypsin revealed a domain structure of DBP that is required for interaction with DNA and that can be disrupted by thermal treatment. Binding to ssDNA, but not to dsDNA, changed the pattern of proteolytic fragments of DBP indicating adjustments in protein structure upon interaction with ssDNA. DBP was capable of unwinding short DNA duplexes and also promoted the renaturation of long complementary strands of ssDNA into duplexes. The unwinding and renaturation activities of DBP, as well as the DNA binding activity, were sensitive to sulfhydryl reagents and were inhibited by oxidation of thiol groups with diamide or by alkylation with N-ethylmaleimide. A high affinity of DBP for ssDNA and its unwinding and renaturation activities confirmed identification of DBP as a member of the SSB/recombinase family. These activities and a tight association with subnuclear structures suggests that DBP is a component of the virogenic stroma that is involved in the processing of replicative intermediates.

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Studies of the Silencing of Baculovirus DNA Binding Protein

Cited by 12 publications

References 25 publications

Transactivator IE1 Is Required for Baculovirus Early Replication Events That Trigger Apoptosis in Permissive and Nonpermissive Cells

Transactivator IE1 Is Required for Baculovirus Early Replication Events That Trigger Apoptosis in Permissive and Nonpermissive Cells

Functional analysis of the putative antiapoptotic genes, p49 and iap4, of Spodoptera litura nucleopolyhedrovirus with RNAi

Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus

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